Shunsuke Kuroki

Epigenetic Regulation of Mouse Sex Determination by the Histone Demethylase Jmjd1a

More Determined Sex Although several transcription factors participate in mammalian sex determination, the contribution from specific epigenetic regulation is just being revealed. Kuroki et al. (p. 1106) show that a JmjC domain–containing protein, Jmjd1a, catalyzes H3K9 demethylation of the Y-linked sex-determining gene Sry in mice to enable its expression above the required threshold level. Ablation of Jmjd1a function results in mouse male-to-female sex reversal, hence not only revealing a mechanism of Sry regulation but also the pivotal role of epigenetic regulation in mammalian sex determination. Histone modification controls mammalian sex determination. Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.

Transforming Growth Factor β-Dependent Sequential Activation of Smad, Bim, and Caspase-9 Mediates Physiological Apoptosis in Gastric Epithelial Cells

ABSTRACT Transforming growth factor β (TGF-β) has been implicated in the maintenance of homeostasis in various organs, including the gastric epithelium. In particular, TGF-β-induced signaling was shown to be required for the differentiation-associated physiological apoptosis of gastric epithelial cells, but its mechanism has not been well understood. In this study, the molecular mechanism of TGF-β-induced apoptosis was analyzed in a human gastric epithelial cell line, SNU16, as an in vitro model. Expression of Smad7 and Bcl-XL, but not viral FLIP, was shown to prevent TGF-β-induced apoptosis, indicating an exclusive requirement of the activation of Smad signaling pathway and mitochondrial dysfunction followed by activation of caspase-9. In addition, treatment with TGF-β induced binding of Bim, a proapoptotic Bcl-2 homology domain 3 (BH3)-only protein, to Bcl-XL, which is dependent on the activation of Smad, and reduction in the expression of Bim by RNA interference decreased the sensitivity to TGF-β-induced apoptosis. Moreover, we found abnormalities in the gastric epithelium of both Bim and caspase-9 knockout mice; these abnormalities were associated with a defect of physiological apoptosis in gastric epithelial cells. These results indicate for the first time that TGF-β is involved in the physiological loss of gastric epithelial cells by activating apoptosis mediated by Smad, Bim, and caspase-9.

Homeoproteins Six1 and Six4 regulate male sex determination and mouse gonadal development

The Y-linked gene Sry regulates mammalian sex determination in bipotential embryonic gonads. Here, we report that the transcription factors Six1 and Six4 are required for male gonadal differentiation. Loss of Six1 and Six4 together, but neither alone, resulted in a male-to-female sex-reversal phenotype in XY mutant gonads accompanied by a failure in Sry activation. Decreased gonadal precursor cell formation at the onset of Sry expression and a gonadal size reduction in both sexes were also found in mutant embryos. Forced Sry transgene expression in XY mutant gonads rescued testicular development but not the initial disruption to precursor growth. Furthermore, we identified two downstream targets of Six1/Six4 in gonadal development, Fog2 (Zfpm2) and Nr5a1 (Ad4BP/Sf1). These two distinct Six1/Six4-regulated pathways are considered to be crucial for gonadal development. The regulation of Fog2 induces Sry expression in male sex determination, and the regulation of Nr5a1 in gonadal precursor formation determines gonadal size.

JMJD1C, a JmjC Domain-Containing Protein, Is Required for Long-Term Maintenance of Male Germ Cells in Mice

ABSTRACT JmjC domain-containing proteins are a class of enzymes responsible for histone demethylation. Previous studies revealed that the JmjC domain-containing protein KDM3A possesses intrinsic demethylase activity toward lysine 9 of histone H3 and plays essential roles in spermiogenesis. In contrast, the biological roles of JMJD1C, a KDM3A homolog in mice, are largely unknown. Here we present the crucial role of JMJD1C in male gametogenesis. Jmjd1c-deficient males became infertile due to the progressive reduction of germ cells after 3 mo of age. Importantly, Jmjd1c-deficient testes frequently contained abnormal tubules lacking developmentally immature germ cells. JMJD1C is most abundantly expressed in undifferentiated spermatogonia in mouse testis. The numbers of ZBTB16-positive spermatogonia and apoptotic germ cells in Jmjd1c-deficient testes decreased and increased in an age-dependent manner, respectively. Our studies demonstrated that JMJD1C contributes to the long-term maintenance of the male germ line.

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Background: Caspase-8 inhibits necrosis by regulating RIP1 and RIP3. Results: Caspase-8 knockdown T-cells simultaneously underwent apoptosis and nonapoptotic cell death. Conclusion: Procaspase-8 prevents T-cells from multiple types of RIP1/3-dependent cell death. Significance: RIP1/3 can regulate caspase-3-dependent apoptosis as well as non-apoptotic cell death. Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.

Rescuing the aberrant sex development of H3K9 demethylase Jmjd1a-deficient mice by modulating H3K9 methylation balance

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance.

Development of a general-purpose method for cell purification using Cre/loxP-mediated recombination.

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general‐purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock‐in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP‐mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1‐Cre‐transgenic (tg) embryos almost equally as efficiently as from Nr5a1‐hCD271‐tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh‐Cre‐tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. genesis 53:387–393, 2015.

FGF2 Has Distinct Molecular Functions from GDNF in the Mouse Germline Niche

Summary Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche.

Epigenetic regulation of mammalian sex determination

Regulation of gene expression without changing the DNA sequence is governed by epigenetic mechanisms. Epigenetic regulation is important for changing chromatin structure in response to environmental cues as well as maintaining chromatin structure after cell division. The epigenetic machinery can reversibly change chromatin function, allowing a wide variety of biological processes in multicellular organisms to be controlled. Epigenetic regulation ensures spatial and temporal accuracy of the expression of developmentally regulated genes. So far, few studies have focused on the relationship between epigenetic regulation and mammalian sex development, despite this being an interesting area of research. Sex development consists of three sequential stages: the undifferentiated stage, gonadal differentiation into testes or ovaries, and differentiation of internal and external genitalia. Some genetic studies have revealed that epigenetic regulation is required for proper gonadal differentiation in mice. Particularly, the epigenetic machinery plays an integral part in sex determination, which is the first step of gonadal differentiation. Mammalian sex determination is triggered by activation of the mammalian sex-determining gene, Sry, in a spatially and temporally accurate manner. Several studies have demonstrated that expression of Sry is controlled not only by specific transcription factors but also by the epigenetic machinery. Here, we focus on the epigenetic regulation of Sry expression.

Combined Loss of JMJD1A and JMJD1B Reveals Critical Roles for H3K9 Demethylation in the Maintenance of Embryonic Stem Cells and Early Embryogenesis

Summary Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome.